Smith-Lemli-Opitz syndrome (SLOS) is a dysmorphology and mental retardation disorder lacking adequate therapy options. It is caused by cholesterol deficiency due to inactivity of 7-dehydrocholesterol reductase (DHCR7). Biochemically, it is characterized by low concentrations of cholesterol (C) in blood and tissues and high concentrations of dehydrocholesterol (DHC). Although mutations in the DHCR7 gene have been well described, the pathogenic development of the disease characteristics is not well understood. Among inherited errors of metabolism, SLOS is relatively frequent, and new diagnostic methods aid its prenatal detection. The long-term objective of this proposal is to develop a basis for the treatment of SLOS. For experiments, gene transfer techniques and mouse models that mimic human SLOS will be used. Three Specific Aims are proposed: (Aim 1) Define the limitations of post-natal, systemic gene therapy. The ratio of DHC/C is an indicator of SLOS severity. So far we have improved, but not completely normalized, this ratio by gene therapy. We now seek to enhance the effectiveness of this gene therapy. Our hypothesis is that the number of liver cells able to express the introduced DHCR7 gene is the limiting factor. We shall test this by varying the dose and type of vector carrying a tagged DHCR7 gene, following the extent of transduction by microscopic identification of cells expressing a tagged enzyme (FLAG-DHCR7), and comparing with DHC/C. (Aim 2) Normalize cholesterol in the CNS by treating newborns. Cholesterol metabolism in the CNS must be treated separately because it is isolated from systemic cholesterol by the blood brain barrier. Gene transfer vector will be injected intrathecally into the cerebrospinal fluid of newborn SLOS mice. At various times brains will be analyzed for sterols, DHCR7 DNA and mRNA, and the distribution of cells expressing FLAG-DHCR7. Sustained normalization of DHC/C in brain will prompt behavioral tests to see if learning, memory and neuromuscular performance are improved. (Aim 3) Correct cholesterol levels during fetal development. Here the emphasis is on preventive treatment and establishing when during development irreversible damage occurs. Biochemical markers, dysmorphic features, and physiological indicators (e.g., survival rates, weight gain and photosensitivity) will all be monitored. If there is significant improvement we shall again explore neuromuscular and behavioral performance. For the three types of treatment, systemic, CNS and fetal, long- term consequences, safety and potential adverse effects will also be monitored. Due to the multiple essential roles of cholesterol, SLOS is a complicated and devastating disorder in spite of its relatively simple genetic origin. The frequency of SLOS in North America is estimated at about 1 in 60,000, although many cases are thought to be never diagnosed because of prenatal lethality or mild phenotype. Through the proposed experiments using gene transfer and SLOS mice, we hope to learn more about the timing and biochemistry of disease development and to establish a basis for eventual treatment in humans. PUBLIC HEALTH RELEVANCE: Smith-Lemli-Opitz syndrome (SLOS) is an inherited condition that is physically and mentally devastating for patients, emotionally devastating for their families and a burden on the health care system. There is no cure, and current treatment of symptoms is inadequate. We are attacking this problem directly by using gene therapy to replace the defective gene that is the root cause of SLOS.